CN113045674A - Human adenovirus five serotype universal antigen epitope fusion protein and preparation and application thereof - Google Patents

Abstract

The invention discloses a human adenovirus five serotype universal antigen epitope fusion protein and preparation and application thereof, relating to the fields of genetic engineering technology, vaccine and diagnostic reagent. Analyzing the amino acid sequences of hexon proteins of type 3, type 7, type 11, type 14 and type 55 of human adenovirus by a computer, comparing and screening out protein fragments with higher homology, connecting the fragments by using two glycines and a serine to form a fusion protein with multiple antigen fragments in series connection, wherein the total length is 411 amino acids, selecting codons preferred by pronucleus, chemically synthesizing the full-length gene of the fusion protein, and expressing and preparing the fusion protein by utilizing a genetic engineering technology. The expressed fusion protein can be used for development of HAdV vaccines, antibodies or antigen detection reagents and the like.

Description

Human adenovirus five serotype universal antigen epitope fusion protein and preparation and application thereof

Technical Field

The invention provides five serotype universal antigen epitope fusion proteins of adenovirus and preparation and application thereof, and relates to the fields of genetic engineering technology, vaccines and diagnostic reagents. The invention connects the 3, 7, 11, 14 and 55 high-homology antigen epitope protein fragments of human adenovirus (HAdV) in series, and prepares the fusion protein containing the strong antigen epitope of five serotype adenoviruses by using the genetic engineering technology. Through computer analysis, the protein fragments of the antigen epitope with higher homology are screened out from the front and middle segments of the hexon protein of the five serotype adenoviruses, and the protein fragments are connected in series by two glycines and one serine to form a multi-antigen epitope fusion protein. The gene sequence of the fusion protein is chemically synthesized, and the fusion protein is expressed by utilizing the gene engineering technology. The expressed fusion protein can be used for detection of the HAdV antibody, development of vaccines and the like.

Background

Human adenovirus, hadv (human adenovirus), is a virus that is highly dangerous to high-density people in kindergartens, schools, and the like. Adenovirus mainly causes respiratory diseases, but can also infect parts such as digestive tract, urinary tract, eyes, cardiac muscle and the like to cause diseases. The adenovirus is double-stranded DNA virus without outer shell, and belongs to the family of adenoviridae, the genome has a total length of 34.7kb, and the capsid has a regular 20-sided structure and a diameter of 80-100 nm. The core consists of double-strand DNA and protein, the outside has nucleocapsid, the upper has 252 particles, and the core consists of 240 hexons and 12 pentamers. Epitopes on the hexon are the criteria for distinguishing between different serotypes and are the sites of the virus most sensitive to immunoselection pressure.

Currently, there are two general methods for detecting adenovirus infection in the laboratory. Firstly, the method is complex, difficult to operate and difficult to type adenovirus by specificity detection of the adenovirus nucleic acid through polymerase chain reaction. And secondly, detecting specific IgM and IgG antibodies in serum by using an enzyme-linked immunosorbent assay. After adenovirus infection, strong immune response can be induced, and specific antibodies can be generated. Typically, IgM production begins in patients 1 week after onset of disease, IgG production begins 7-10 days later, and then gradually increases. Specific antibodies in serum can therefore be detected using specific antigens. However, currently, only one type of adenovirus antibody can be detected by one antigen, so that multiple antigens are needed to improve the antibody detection rate when detecting the HAdV antibody in serum, confirm the infection type, improve the detection cost, and still have a certain false negative probability. The development of multi-epitope gene recombinant antigen is the development direction for establishing adenovirus specific antibody rapid detection reagent and the development direction for developing vaccine.

Disclosure of Invention

The invention aims to provide a human adenovirus five-serotype universal antigen epitope fusion protein aiming at the defects, wherein the hexon protein of the human adenovirus (HAdV) shell has a plurality of epitopes and is the most sensitive part of a virosome to immunoselection pressure, and an organism is induced to generate a plurality of specific antibodies, so the protein is an ideal protein for developing a human adenovirus (HAdV) antibody detection reagent by using an antigen.

The amino acid sequence of adenovirus hexon protein is analyzed by a computer, five protein fragments containing universal strong antigen epitope of the adenovirus serotype are respectively screened out, and the protein fragments are connected in series by two glycins and one serine to form a multi-antigen epitope fusion protein. The gene sequence of the fusion protein is chemically synthesized, and the fusion protein is expressed by utilizing the gene engineering technology. The expressed fusion protein can be used for detecting HAdV antibody or antigen, developing vaccine and the like.

The invention relates to a chemical synthesis human adenovirus (HAdV), which prepares fusion protein containing five types of universal adenovirus antigen epitopes by using gene engineering technology and using the gene sequences of universal antigen epitope fusion proteins of adenovirus types 3, 7, 11, 14 and 55. The amino acid sequences of the hexon proteins of types 3, 7, 11, 14 and 55 of the human adenovirus are analyzed by a computer, the first 135 amino acid fragments with higher homology are screened out, and the differential fragments of the first 135 amino acids with higher homology of the five serotype adenoviruses contain strong antigen epitopes. Therefore, the first 135 amino acid fragments of the adenovirus hexon proteins of the five serotypes are connected by two glycines and one serine and are connected in series to form a multi-epitope fusion protein with 411 amino acids in total length. The codon preferred by prokaryote is selected, the fusion protein is translated into corresponding nucleotide sequence, and the full-length gene sequence is chemically synthesized. The fusion protein is expressed and purified by using a genetic engineering technology. The fusion protein can be used for detecting vaccines and HAdV antibodies or antigens, and can be used for immune preparation of anti-HAdV monoclonal antibodies, anti-HAdV polyclonal antibodies and the like.

Human adenovirus (HAdV) five serotype universal antigen epitope fusion protein and preparation and application thereof are implemented by adopting the following steps:

1. screening five serotypes of human adenovirus general antigen epitope and chemically synthesizing fusion protein gene fragment:

the amino acid sequences of hexon proteins of type 3, 7, 11, 14 and 55 of human adenovirus are analyzed by using software such as ANTHWIN and the like, and protein fragments with higher homology in the hexon proteins of five serotype adenovirus are screened out. The protein fragments are connected in series by two glycines and one serine to form a multi-antigen epitope fusion protein. The codon preferred by prokaryotes is selected and the fusion protein is translated into the corresponding nucleotide sequence. At the 5' end of the fusion protein gene segment is increasedBamH I cleavage site (underlined), addition of a stop codon at the 3' end andXho i cleavage site (underlined). Making it easy to clone the synthetic gene fragment into plasmid pET-28a (+)BamH I andXho i, enzyme cutting site. The full-length gene sequence of the fusion protein is chemically synthesized.

The amino acid sequence of the epitope of adenovirus type 3:

aa at positions 1 to 135:

Met Ala Thr Pro Ser Met Met Pro Gln Trp Ala Tyr Met His Ile

Ala Gly Gln Asp Ala Ser Glu Tyr Leu Ser Pro Gly Leu Val Gln

Phe Ala Arg Ala Thr Asp Thr Tyr Phe Ser Met Gly Asn Lys Phe

Arg Asn Pro Thr Val Ala Pro Thr His Asp Val Thr Thr Asp Arg

Ser Gln Arg Leu Met Leu Arg Phe Val Pro Val Asp Arg Glu Asp

Asn Thr Tyr Ser Tyr Lys Val Arg Tyr Thr Leu Ala Val Gly Asp

Asn Arg Val Leu Asp Met Ala Ser Thr Phe Phe Asp Ile Arg Gly

Val Leu Asp Arg Gly Pro Ser Phe Lys Pro Tyr Ser Gly Thr Ala

Tyr Asn Ser Leu Ala Pro Lys Gly Ala Pro Asn Thr Ser Gln Trp；

the screened adenovirus type 7 epitope amino acid sequence:

aa at positions 1 to 135:

Met Ala Thr Pro Ser Met Met Pro Gln Trp Ala Tyr Met His Ile

Ala Gly Gln Asp Ala Ser Glu Tyr Leu Ser Pro Gly Leu Val Gln

Phe Ala Arg Ala Thr Asp Thr Tyr Phe Ser Met Gly Asn Lys Phe

Arg Asn Pro Thr Val Ala Pro Thr His Asp Val Thr Thr Asp Arg

Ser Gln Arg Leu Met Leu Arg Phe Val Pro Val Asp Arg Glu Asp

Asn Thr Tyr Ser Tyr Lys Val Arg Tyr Thr Leu Ala Val Gly Asp

Asn Arg Val Leu Asp Met Ala Ser Thr Phe Phe Asp Ile Arg Gly

Val Leu Asp Arg Gly Pro Ser Phe Lys Pro Tyr Ser Gly Thr Ala

Tyr Asn Ser Leu Ala Pro Lys Gly Ala Pro Asn Thr Ser Gln Trp；

the screened adenovirus type 11 epitope amino acid sequence:

aa at positions 1 to 135:

Met Ala Thr Pro Ser Met Leu Pro Gln Trp Ala Tyr Met His Ile

Ala Gly Gln Asp Ala Ser Glu Tyr Leu Ser Pro Gly Leu Val Gln

Phe Ala Arg Ala Thr Asp Thr Tyr Phe Asn Leu Gly Asn Lys Phe

Arg Asn Pro Thr Val Ala Pro Thr His Asp Val Thr Thr Asp Arg

Ser Gln Arg Leu Met Leu Arg Phe Val Pro Val Asp Arg Glu Asp

Asn Thr Tyr Ser Tyr Lys Val Arg Tyr Thr Leu Ala Val Gly Asp

Asn Arg Val Leu Asp Met Ala Ser Thr Phe Phe Asp Ile Arg Gly

Val Leu Asp Arg Gly Pro Ser Phe Lys Pro Tyr Ser Gly Thr Ala

Tyr Asn Ser Leu Ala Pro Lys Gly Ala Pro Asn Ala Ser Gln Trp；

the screened adenovirus type 14 epitope amino acid sequence:

aa at positions 1 to 135:

Met Ala Thr Pro Ser Met Leu Pro Gln Trp Ala Tyr Met His Ile

Ala Gly Gln Asp Ala Ser Glu Tyr Leu Ser Pro Gly Leu Val Gln

Phe Ala Arg Ala Thr Asp Thr Tyr Phe Asn Leu Gly Asn Lys Phe

Arg Asn Pro Thr Val Ala Pro Thr His Asp Val Thr Thr Asp Arg

Ser Gln Arg Leu Met Leu Arg Phe Val Pro Val Asp Arg Glu Asp

Asn Thr Tyr Ser Tyr Lys Val Arg Tyr Thr Leu Ala Val Gly Asp

Asn Arg Val Leu Asp Met Ala Ser Thr Phe Phe Asp Ile Arg Gly

Val Leu Asp Arg Gly Pro Ser Phe Lys Pro Tyr Ser Gly Thr Ala

Tyr Asn Ser Leu Ala Pro Lys Gly Ala Pro Asn Ala Ser Gln Trp；

the screened adenovirus type 55 epitope amino acid sequence:

aa at positions 1 to 135:

Met Ala Thr Pro Ser Met Leu Pro Gln Trp Ala Tyr Met His Ile

Ala Gly Gln Asp Ala Ser Glu Tyr Leu Ser Pro Gly Leu Val Gln

Phe Ala Arg Ala Thr Asp Thr Tyr Phe Asn Leu Gly Asn Lys Phe

Arg Asn Pro Thr Val Ala Pro Thr His Asp Val Thr Thr Asp Arg

Ser Gln Arg Leu Met Leu Arg Phe Val Pro Val Asp Arg Glu Asp

Asn Thr Tyr Ser Tyr Lys Val Arg Tyr Thr Leu Ala Val Gly Asp

Asn Arg Val Leu Asp Met Ala Ser Thr Phe Phe Asp Ile Arg Gly

Val Leu Asp Arg Gly Pro Ser Phe Lys Pro Tyr Ser Gly Thr Ala

Tyr Asn Ser Leu Ala Pro Lys Gly Ala Pro Asn Thr Ser Gln Trp；

fusion protein amino acid sequence (411 aa):

Met Ala Thr Pro Ser Met Met Pro Gln Trp Ala Tyr Met His Ile

Ala Gly Gln Asp Ala Ser Glu Tyr Leu Ser Pro Gly Leu Val Gln

Phe Ala Arg Ala Thr Asp Thr Tyr Phe Ser Met Gly Asn Lys Phe

Arg Asn Pro Thr Val Ala Pro Thr His Asp Val Thr Thr Asp Arg

Ser Gln Arg Leu Met Leu Arg Phe Val Pro Val Asp Arg Glu Asp

Asn Thr Tyr Ser Tyr Lys Val Arg Tyr Thr Leu Ala Val Gly Asp

Asn Arg Val Leu Asp Met Ala Ser Thr Phe Phe Asp Ile Arg Gly

Val Leu Asp Arg Gly Pro Ser Phe Lys Pro Tyr Ser Gly Thr Ala

Tyr Asn Ser Leu Ala Pro Lys Gly Ala Pro Asn Thr Ser Gln Trp

Gly Gly Ser Met Ala Thr Pro Ser Met Leu Pro Gln Trp Ala Tyr

Met His Ile Ala Gly Gln Asp Ala Ser Glu Tyr Leu Ser Pro Gly

Leu Val Gln Phe Ala Arg Ala Thr Asp Thr Tyr Phe Asn Leu Gly

Asn Lys Phe Arg Asn Pro Thr Val Ala Pro Thr His Asp Val Thr

Thr Asp Arg Ser Gln Arg Leu Met Leu Arg Phe Val Pro Val Asp

Arg Glu Asp Asn Thr Tyr Ser Tyr Lys Val Arg Tyr Thr Leu Ala

Val Gly Asp Asn Arg Val Leu Asp Met Ala Ser Thr Phe Phe Asp

Ile Arg Gly Val Leu Asp Arg Gly Pro Ser Phe Lys Pro Tyr Ser

Gly Thr Ala Tyr Asn Ser Leu Ala Pro Lys Gly Ala Pro Asn Thr

Ser Gln Trp Gly Gly Ser Met Ala Thr Pro Ser Met Leu Pro Gln

Trp Ala Tyr Met His Ile Ala Gly Gln Asp Ala Ser Glu Tyr Leu

Ser Pro Gly Leu Val Gln Phe Ala Arg Ala Thr Asp Thr Tyr Phe

Asn Leu Gly Asn Lys Phe Arg Asn Pro Thr Val Ala Pro Thr His

Asp Val Thr Thr Asp Arg Ser Gln Arg Leu Met Leu Arg Phe Val

Pro Val Asp Arg Glu Asp Asn Thr Tyr Ser Tyr Lys Val Arg Tyr

Thr Leu Ala Val Gly Asp Asn Arg Val Leu Asp Met Ala Ser Thr

Phe Phe Asp Ile Arg Gly Val Leu Asp Arg Gly Pro Ser Phe Lys

Pro Tyr Ser Gly Thr Ala Tyr Asn Ser Leu Ala Pro Lys Gly Ala

Pro Asn Thr Ser Gln Trp；

DNA sequence of chemically synthesized fusion protein (1248 bp)

GGA TCC ATG GCT ACA CCC TCA ATG ATG CCA CAA TGG GCG TAT ATG CAC ATT GCC GGT CAA GAC GCG TCC GAA TAC CTC TCT CCG GGT CTG GTT CAA TTT GCC AGA GCC ACC GAC ACC TAT TTC AGC ATG GGT AAT AAG TTC CGT AAT CCG ACG GTT GCA CCG ACG CAT GAT GTT ACC ACA GAT CGT AGC CAA CGT CTG ATG CTG CGC TTT GTT CCG GTG GAC CGC GAG GAC AAC ACC TAC TCC TAC AAG GTG CGT TAC ACC CTG GCG GTC GGT GAC AAC CGT GTC CTG GAT ATG GCG TCT ACC TTT TTC GAT ATT CGT GGT GTA CTT GAC CGC GGA CCG AGC TTC AAA CCG TAT AGC GGC ACC GCA TAC AAC AGC CTG GCG CCG AAA GGC GCA CCA AAT ACG AGC CAA TGG GGT GGC AGC ATG GCT ACT CCG AGC ATG TTA CCG CAG TGG GCG TAT ATG CAC ATC GCA GGC CAG GAT GCA AGC GAA TAT CTG TCT CCG GGT CTG GTG CAG TTC GCG CGT GCT ACC GAC ACC TAC TTC AAC CTG GGC AAC AAA TTC CGC AAC CCG ACC GTT GCG CCG ACG CAC GAC GTG ACG ACC GAC CGT TCC CAA CGT CTG ATG CTG CGT TTC GTG CCG GTC GAT CGT GAG GAT AAC ACG TAC TCG TAC AAA GTA CGT TAT ACC TTG GCT GTG GGC GAC AAC CGT GTT CTC GAT ATG GCG AGT ACC TTT TTT GAT ATC CGC GGC GTA TTG GAC CGC GGT CCG TCT TTC AAG CCG TAC TCT GGC ACT GCG TAT AAC AGC CTG GCT CCG AAG GGT GCA CCG AAT ACT TCA CAG TGG GGC GGT AGC ATG GCC ACC CCG AGC ATG CTG CCT CAG TGG GCA TAC ATG CAT ATC GCG GGT CAG GAT GCT TCG GAG TAC TTG AGC CCT GGT TTG GTT CAG TTT GCG CGG GCT ACC GAC ACC TAT TTT AAC CTG GGT AAT AAA TTT CGT AAT CCG ACC GTT GCG CCG ACC CAT GAT GTG ACC ACC GAT CGA TCC CAG CGT TTG ATG CTG CGT TTC GTG CCG GTT GAC CGC GAA GAT AAC ACT TAT TCC TAT AAG GTG CGC TAC ACC CTT GCG GTT GGT GAC AAC CGC GTC CTG GAC ATG GCT TCC ACC TTT TTC GAC ATT CGT GGC GTG TTG GAC AGA GGC CCG AGT TTT AAG CCG TAT AGC GGT ACG GCC TAC AAT TCC CTG GCG CCA AAA GGC GCC CCA AAT GCA AGC CAA TGG TAA CTC GAG。

2. Construction of recombinant plasmid expressing five serotypes of human adenovirus universal epitope fusion protein:

extraction of plasmid pET-28a (+)(Novagen, USA) fromBamH I andXho i, carrying out double enzyme digestion, recovering the digested plasmid large fragment after electrophoresis, and dissolving in deionized water. By usingBamH I andXho i, performing double enzyme digestion chemical synthesis on five serotype universal antigen epitope fusion protein gene segments of the human adenovirus, and dissolving in deionized water after electrophoretic recovery. Inserting the above-mentioned DNA fragment after enzyme digestion into vector pET-28a (+)BamH I andXho and five types of fusion proteins of adenovirus universal antigen epitopes are expressed among the I sites.

3. Screening and identifying recombinant plasmids:

the recombinant plasmid was transformed into E.coli BL21(DE3), coated with LB plate containing kanamycin (60. mu.g/mL), and left overnight at 37 ℃. Randomly picking transformed colony and control bacteria (plasmid pET-28a (+) transformed bacteria) the next day, extracting plasmid, and usingBamH I andXho i, double enzyme digestion verification, and a result of 1.0% agarose gel electrophoresis shows that the 1248bp target gene fragment is cut off. Meanwhile, DNA sequence analysis is carried out on the plasmid containing the gene segment, and the sequence analysis proves that the recombinant plasmid contains five types of adenovirus antigen epitope gene segments which are connected together and has completely correct sequence.

The constructed recombinant plasmid expresses fusion protein containing five serotype adenovirus antigen epitopes, the total length is 445 amino acids, each segment is connected by two glycin and one serine, and the amino acid sequence is as follows:

Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val

Pro Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Glu Glu Met

Gly Arg Gly Ser Met Ala Thr Pro Ser Met Met Pro Gln Trp Ala

Tyr Met His Ile Ala Gly Gln Asp Ala Ser Glu Tyr Leu Ser Pro

Gly Leu Val Gln Phe Ala Arg Ala Thr Asp Thr Tyr Phe Ser Met

Gly Asn Lys Phe Arg Asn Pro Thr Val Ala Pro Thr His Asp Val

Thr Thr Asp Arg Ser Gln Arg Leu Met Leu Arg Phe Val Pro Val

Asp Arg Glu Asp Asn Thr Tyr Ser Tyr Lys Val Arg Tyr Thr Leu

Ala Val Gly Asp Asn Arg Val Leu Asp Met Ala Ser Thr Phe Phe

Asp Ile Arg Gly Val Leu Asp Arg Gly Pro Ser Phe Lys Pro Tyr

Ser Gly Thr Ala Tyr Asn Ser Leu Ala Pro Lys Gly Ala Pro Asn

Thr Ser Gln Trp Gly Gly Ser Met Ala Thr Pro Ser Met Leu Pro

Gln Trp Ala Tyr Met His Ile Ala Gly Gln Asp Ala Ser Glu Tyr

Leu Ser Pro Gly Leu Val Gln Phe Ala Arg Ala Thr Asp Thr Tyr

Phe Asn Leu Gly Asn Lys Phe Arg Asn Pro Thr Val Ala Pro Thr

His Asp Val Thr Thr Asp Arg Ser Gln Arg Leu Met Leu Arg Phe

Val Pro Val Asp Arg Glu Asp Asn Thr Tyr Ser Tyr Lys Val Arg

Tyr Thr Leu Ala Val Gly Asp Asn Arg Val Leu Asp Met Ala Ser

Thr Phe Phe Asp Ile Arg Gly Val Leu Asp Arg Gly Pro Ser Phe

Lys Pro Tyr Ser Gly Thr Ala Tyr Asn Ser Leu Ala Pro Lys Gly

Ala Pro Asn Thr Ser Gln Trp Gly Gly Ser Met Ala Thr Pro Ser

Met Leu Pro Gln Trp Ala Tyr Met His Ile Ala Gly Gln Asp Ala

Ser Glu Tyr Leu Ser Pro Gly Leu Val Gln Phe Ala Arg Ala Thr

Asp Thr Tyr Phe Asn Leu Gly Asn Lys Phe Arg Asn Pro Thr Val

Ala Pro Thr His Asp Val Thr Thr Asp Arg Ser Gln Arg Leu Met

Leu Arg Phe Val Pro Val Asp Arg Glu Asp Asn Thr Tyr Ser Tyr

Lys Val Arg Tyr Thr Leu Ala Val Gly Asp Asn Arg Val Leu Asp

Met Ala Ser Thr Phe Phe Asp Ile Arg Gly Val Leu Asp Arg Gly

Pro Ser Phe Lys Pro Tyr Ser Gly Thr Ala Tyr Asn Ser Leu Ala

Pro Lys Gly Ala Pro Asn Thr Ser Gln Trp。

4. screening and identifying the engineering bacteria expressing the fusion protein:

the positive transformant containing the recombinant plasmid is inoculated into a test tube containing 3mL of LB culture medium (containing kanotoxin 60 mu g/mL), the shaking culture is carried out for 3h at 37 ℃, IPTG is added to the final concentration of 0.5mmol/L, the shaking culture induction is continued for 4h, the thalli are collected by centrifugation and are subjected to SDS-PAGE detection, the recombinant expresses HAdV fusion protein with the relative molecular weight of 50kD, and a control bacterium BL21(DE3) does not have the protein band.

5. Purification of five serotype universal antigen epitope fusion proteins of human adenovirus:

1) ultrasonic lysis of five serotype universal antigen epitope fusion protein engineering bacteria of human adenovirus

The engineering bacteria inducing expression of the fusion protein are centrifuged (8000 rpm, 15min and 4 ℃) to collect bacteria, the bacteria are suspended in bacterial lysate (20 mmol/L PB pH7.4 and 5% glycerol) with the volume of 1/10 of original culture solution, the bacteria are broken by ultrasonic in ice bath, the precipitate is collected by centrifugation, and the precipitate is the expressed adenovirus epitope fusion protein inclusion body.

2) Denaturation of five serotype universal epitope fusion proteins of human adenovirus

Resuspending and washing the inclusion body by 30ml of inclusion body washing liquor, wherein the inclusion body washing liquor is 1xPBS pH7.4 and 1% Triton X-100, ultrasonically suspending the inclusion body under the ice bath condition until all precipitates are suspended, and centrifugally collecting the precipitated inclusion body. And (3) carrying out heavy suspension dissolution on the inclusion bodies by using 8 mol/L urea and 1xPBS (x-PbS) pH7.4 denaturing solution, and carrying out ultrasonic crushing under an ice bath condition until the inclusion bodies are completely dissolved. The supernatant was collected by centrifugation at 8000 rpm, 15min and 4 ℃.

3) Purifying by nickel column affinity chromatography

Washing Ni-NTA Resin with 1xPBS pH7.4, removing excessive 20% ethanol, filtering the supernatant collected by centrifugation with 0.22 μm filter membrane, removing impurities, mixing with Ni-NTA Resin to allow adenovirus universal epitope fusion protein to bind with Ni-NTA Resin, and stirring with glass rod for 1.5 h. And then loading the mixed solution on a column, washing by using a balance solution, sequentially using elution proteins of 20, 40, 100, 200 and 500 mmol/L imidazole eluent containing 8 mol/L urea, collecting each elution peak protein, detecting each peak protein by using 12% SDS-PAGE electrophoresis, and determining which elution peak contains the expressed adenovirus epitope fusion protein. The result shows that the peak of the imidazole elution protein of 100 mmol/L is the adenovirus epitope fusion protein.

4) Renaturation of five serotype universal antigen epitope fusion protein of human adenovirus

The denatured fusion protein obtained by purification is sequentially placed in PBS pH7.4 dialysate containing 6, 4, 3, 2, 1, 0 mol/L urea for sequential dialysis renaturation.

6. The expressed human adenovirus five serotype universal antigen epitope fusion protein is used for vaccine, HAdV antibody or antigen detection and immune preparation of anti-HAdV monoclonal antibody and polyclonal antibody.

7. The high homology protein fragments of the hexon proteins of the five serotype adenoviruses are connected and expressed and prepared in the form of fusion protein.

8. By gene recombination technology, yeast cells, insect cells, mammal cells and transgenic animals and plants are used for carrying out recombination expression to prepare the adenovirus epitope fusion protein.

Description of english abbreviations: HAdV (human adenovirus), EDTA tetramethylethylenediamine; IPTG: isopropyl thiogalactoside; DTT: dithiothreitol; SDS (sodium dodecyl sulfate): sodium dodecyl sulfonate; PAGE: performing polyacrylamide gel electrophoresis; PB: phosphate buffer; kD: kilodaltons.

Compared with the prior art, the invention has the advantages that:

1. the five serotype adenovirus universal antigen epitope protein fragments are fused and expressed, and are more convenient and economical than the single protein fragments. When the fusion protein is used as the antigen to detect the anti-HAdV antibody, the protein fragments do not need to be prepared respectively, and the use proportion of the antigens does not need to be adjusted, so the application is convenient and the cost is low.

2. At present, adenovirus genetic engineering subunit vaccines do not exist, and need to be developed urgently. The fusion protein containing various adenovirus hexon antigen epitopes is prepared by using gene engineering technology expression, and a foundation is laid for developing gene engineering vaccines. The genetic engineering vaccine has the advantages of safety and low cost.

Drawings

The invention will be further explained with reference to the drawings, in which:

FIG. 1 shows the results of analysis of epitopes of hexon protein types 3, 7, 11, 14 and 55 of adenovirus by molecular biology software. The results show that the homology of the first 135 amino acids of the hexon protein of the adenovirus is higher.

FIG. 2 is a graph of the prediction of the first 135 amino acid B-cell epitopes of the hexon protein of five serotypes of adenovirus.

FIG. 3 is a flow chart of recombinant plasmid construction for expression of five serotype adenovirus epitope fusion proteins.

FIG. 4 shows the SDS-PAGE analysis of recombinant bacteria expressing adenovirus epitope fusion proteins. The constructed recombinant plasmid is transformed into escherichia coli, and SDS-PAGE electrophoresis images of high-yield strains are screened after single strains are selected. M: protein marker; yin: negative control bacteria containing pET-28a (+) plasmid; 1-6: five serotypes of human adenovirus are recombinant bacteria of the universal antigen epitope fusion protein, and six recombinants all express the fusion protein with the relative molecular weight of 50kDa, namely the position marked by an arrow in the figure.

FIG. 5 shows SDS-PAGE analysis of five serotypes of human adenovirus expressing universal epitope fusion protein after purification and renaturation. M: a protein Marker; 1: the concentration of the purified adenovirus epitope fusion protein is 0.5mg/mL, and the relative molecular weight is 50 kDa; 2: BSA at a concentration of 0.5 mg/mL.

FIG. 6 shows ELISA results of antiserum titer of five serotypes of human adenovirus universal epitope fusion protein.

FIG. 7 shows the comparison analysis of the cellular immune ELISA results of five serotype universal antigen epitope fusion proteins of human adenovirus with different immune adjuvants for relieving summer heat.

FIG. 8 shows the results of ELISA detection of five serotypes of human adenovirus fusion protein.

FIG. 9 shows the specificity of test paper for detecting adenovirus antibody, which is developed by using five serotype universal antigen epitope fusion proteins of human adenovirus as antigens.

Detailed Description

Detailed description of embodiments of the invention:

analysis, gene synthesis and expression of adenovirus hexon protein epitope

The amino acid sequences of adenovirus type 3, 7, 11, 14 and 55 hexon proteins are analyzed by a computer, and protein fragments with higher homology are respectively screened out. The first 135 amino acids of the hexon protein of the five serotype adenoviruses are respectively connected by two glycin and one serine and are connected in series to form a multi-epitope fusion protein. The codon preferred by prokaryotes is selected and the fusion protein is translated into the corresponding nucleotide sequence. The full-length gene sequence of the fusion protein is chemically synthesized and cloned into a plasmid pET-28a (+)BamH I / Xho I site, expressing adenovirus epitope fusion protein. The recombinant plasmid is transformed into escherichia coli BL21(DE3), and the engineering bacteria for efficiently expressing the adenovirus epitope fusion protein is obtained by screening.

Materials and methods

1. Strains and plasmids:

the host strain BL21(DE3) and the expression vector pET-28a (+) are products of Novagen company in the United states.

2. Molecular biological reagents:

restriction enzymeBamH I、Xho I. And T4 DNA ligase is TaKaRa. The plasmid purification kit and the kit for recovering DNA fragments from agarose gel are products of TaKaRa company. DTT and IPTG are products of TaKaRa company. Other reagents are imported or domestic analytical pure reagents.

3. Synthesis of gene fragment:

synthesis was aided by TaKaRa Inc., Dalian.

4. The gene cloning method comprises the following steps:

carrying out enzyme digestion, connection and electrophoresis on DNA; extracting and transforming plasmids; general molecular cloning methods such as SDS-PAGE analysis of proteins are carried out by conventional methods. Other kits were run as described.

5. DNA sequence analysis:

the plasmid was purified using a plasmid purification kit from TaKaRa and sequenced using a DNA full-automatic sequencer.

Results

Screening of epitope of HAdV hexon protein and chemical synthesis of gene fragment thereof:

the amino acid sequences of hexon type 3, 7, 11, 14 and 55 adenovirus proteins were analyzed in silico using ANTHWIN software and found to have high homology over the first 135 amino acids. The first 135 amino acids of hexon protein of each type of adenovirus are connected by two glycines and a serine and are connected in series to form a multi-epitope fusion protein. The codon preferred by prokaryotes is selected and the fusion protein is translated into the corresponding nucleotide sequence. At the 5' end of the fusion protein gene sequence is increasedBamH I cleavage site, increased at the 3' endXho I enzyme cutting site. The full-length gene sequence of the fusion protein is chemically synthesized.

Fusion protein amino acid sequence (411 aa):

Met Ala Thr Pro Ser Met Met Pro Gln Trp Ala Tyr Met His Ile

Ala Gly Gln Asp Ala Ser Glu Tyr Leu Ser Pro Gly Leu Val Gln

Phe Ala Arg Ala Thr Asp Thr Tyr Phe Ser Met Gly Asn Lys Phe

Arg Asn Pro Thr Val Ala Pro Thr His Asp Val Thr Thr Asp Arg

Ser Gln Arg Leu Met Leu Arg Phe Val Pro Val Asp Arg Glu Asp

Asn Thr Tyr Ser Tyr Lys Val Arg Tyr Thr Leu Ala Val Gly Asp

Asn Arg Val Leu Asp Met Ala Ser Thr Phe Phe Asp Ile Arg Gly

Val Leu Asp Arg Gly Pro Ser Phe Lys Pro Tyr Ser Gly Thr Ala

Tyr Asn Ser Leu Ala Pro Lys Gly Ala Pro Asn Thr Ser Gln Trp

Gly Gly Ser Met Ala Thr Pro Ser Met Leu Pro Gln Trp Ala Tyr

Met His Ile Ala Gly Gln Asp Ala Ser Glu Tyr Leu Ser Pro Gly

Leu Val Gln Phe Ala Arg Ala Thr Asp Thr Tyr Phe Asn Leu Gly

Asn Lys Phe Arg Asn Pro Thr Val Ala Pro Thr His Asp Val Thr

Thr Asp Arg Ser Gln Arg Leu Met Leu Arg Phe Val Pro Val Asp

Arg Glu Asp Asn Thr Tyr Ser Tyr Lys Val Arg Tyr Thr Leu Ala

Val Gly Asp Asn Arg Val Leu Asp Met Ala Ser Thr Phe Phe Asp

Ile Arg Gly Val Leu Asp Arg Gly Pro Ser Phe Lys Pro Tyr Ser

Gly Thr Ala Tyr Asn Ser Leu Ala Pro Lys Gly Ala Pro Asn Thr

Ser Gln Trp Gly Gly Ser Met Ala Thr Pro Ser Met Leu Pro Gln

Trp Ala Tyr Met His Ile Ala Gly Gln Asp Ala Ser Glu Tyr Leu

Ser Pro Gly Leu Val Gln Phe Ala Arg Ala Thr Asp Thr Tyr Phe

Asn Leu Gly Asn Lys Phe Arg Asn Pro Thr Val Ala Pro Thr His

Asp Val Thr Thr Asp Arg Ser Gln Arg Leu Met Leu Arg Phe Val

Pro Val Asp Arg Glu Asp Asn Thr Tyr Ser Tyr Lys Val Arg Tyr

Thr Leu Ala Val Gly Asp Asn Arg Val Leu Asp Met Ala Ser Thr

Phe Phe Asp Ile Arg Gly Val Leu Asp Arg Gly Pro Ser Phe Lys

Pro Tyr Ser Gly Thr Ala Tyr Asn Ser Leu Ala Pro Lys Gly Ala

Pro Asn Thr Ser Gln Trp；

DNA sequence of chemically synthesized fusion protein (1248 bp)

GGA TCC ATG GCT ACA CCC TCA ATG ATG CCA CAA TGG GCG TAT ATG CAC ATT GCC GGT CAA GAC GCG TCC GAA TAC CTC TCT CCG GGT CTG GTT CAA TTT GCC AGA GCC ACC GAC ACC TAT TTC AGC ATG GGT AAT AAG TTC CGT AAT CCG ACG GTT GCA CCG ACG CAT GAT GTT ACC ACA GAT CGT AGC CAA CGT CTG ATG CTG CGC TTT GTT CCG GTG GAC CGC GAG GAC AAC ACC TAC TCC TAC AAG GTG CGT TAC ACC CTG GCG GTC GGT GAC AAC CGT GTC CTG GAT ATG GCG TCT ACC TTT TTC GAT ATT CGT GGT GTA CTT GAC CGC GGA CCG AGC TTC AAA CCG TAT AGC GGC ACC GCA TAC AAC AGC CTG GCG CCG AAA GGC GCA CCA AAT ACG AGC CAA TGG GGT GGC AGC ATG GCT ACT CCG AGC ATG TTA CCG CAG TGG GCG TAT ATG CAC ATC GCA GGC CAG GAT GCA AGC GAA TAT CTG TCT CCG GGT CTG GTG CAG TTC GCG CGT GCT ACC GAC ACC TAC TTC AAC CTG GGC AAC AAA TTC CGC AAC CCG ACC GTT GCG CCG ACG CAC GAC GTG ACG ACC GAC CGT TCC CAA CGT CTG ATG CTG CGT TTC GTG CCG GTC GAT CGT GAG GAT AAC ACG TAC TCG TAC AAA GTA CGT TAT ACC TTG GCT GTG GGC GAC AAC CGT GTT CTC GAT ATG GCG AGT ACC TTT TTT GAT ATC CGC GGC GTA TTG GAC CGC GGT CCG TCT TTC AAG CCG TAC TCT GGC ACT GCG TAT AAC AGC CTG GCT CCG AAG GGT GCA CCG AAT ACT TCA CAG TGG GGC GGT AGC ATG GCC ACC CCG AGC ATG CTG CCT CAG TGG GCA TAC ATG CAT ATC GCG GGT CAG GAT GCT TCG GAG TAC TTG AGC CCT GGT TTG GTT CAG TTT GCG CGG GCT ACC GAC ACC TAT TTT AAC CTG GGT AAT AAA TTT CGT AAT CCG ACC GTT GCG CCG ACC CAT GAT GTG ACC ACC GAT CGA TCC CAG CGT TTG ATG CTG CGT TTC GTG CCG GTT GAC CGC GAA GAT AAC ACT TAT TCC TAT AAG GTG CGC TAC ACC CTT GCG GTT GGT GAC AAC CGC GTC CTG GAC ATG GCT TCC ACC TTT TTC GAC ATT CGT GGC GTG TTG GAC AGA GGC CCG AGT TTT AAG CCG TAT AGC GGT ACG GCC TAC AAT TCC CTG GCG CCA AAA GGC GCC CCA AAT GCA AGC CAA TGG TAA CTC GAG。

2. Construction of five serotype universal antigen epitope fusion protein recombinant plasmids of human adenovirus:

extraction of plasmid pET-28a (+) (Novagen, USA)By usingBamH I andXho i, carrying out double enzyme digestion, recovering the digested plasmid large fragment after electrophoresis, and dissolving in deionized water. By usingBamH I andXho i, performing double enzyme digestion chemical synthesis on five serotype universal antigen epitope fusion protein gene segments of the human adenovirus, and dissolving in deionized water after electrophoretic recovery. Inserting the above-mentioned DNA fragment after enzyme digestion into vector pET-28a (+)BamH I andXho among I sites, a fusion protein containing five serotype adenovirus antigen epitopes is expressed. The construction flow is shown in FIG. 3.

3. Screening and identifying recombinant plasmids:

the recombinant plasmid ligated in the above step was transformed into E.coli BL21(DE3), and the transformed product was spread on LB plate containing 60. mu.g/mL kanamycin and left overnight at 37 ℃. The next day, 5 transformant colonies were randomly selected, inoculated into tubes containing 4 mL of liquid LB medium (containing 60. mu.g/mL kanamycin), subjected to shake culture at 37 ℃ for 6 hours, and centrifuged to collect 1mL of bacterial solution. Respectively suspending thallus with 50 μ L deionized water, boiling in water for 5min, centrifuging at 4 deg.C and 12000rpm for 5min, taking 1 μ L supernatant containing plasmid as PCR template, and PCR amplifying positive recombinant plasmid containing fusion protein gene fragment to obtain gene fragment with length of 1248 bp. The PCR reaction concentration is: 1 μ L of plasmid template, 1 μ L of each of the upstream and downstream primers, 5.0 μ L of 10 × Buffer, 4.0 μ L of 2.5 mmol/L dNTP, 0.5 μ L (2.5U) of Taq DNA polymerase, and 37.5 μ L of deionized water, in a total volume of 50 μ L. The amplification conditions were: 30 cycles of 94 ℃ for 30 seconds, 60 ℃ for 30 seconds, 72 ℃ for 60 seconds; final extension at 72 ℃ for 7 min. Taking 5 mu L of PCR amplification product, detecting by using 1.2% Agarose gel, and amplifying a 1248bp target gene fragment by a transformant. It is proved that all the genes contain tandem genes of five serotype adenovirus epitope gene segments.

Extracting plasmid of transformant, determining serial gene sequence of five serotype adenovirus epitope gene segments in the plasmid, and DNA sequence analysis proves that the recombinant plasmid contains five serotype adenovirus epitope gene segments with complete correct sequence.

The constructed recombinant plasmid expresses fusion protein containing five serotype adenovirus antigen epitopes, the total length is 445 amino acids, the fragments are connected by two glycin and one serine, and the amino acid sequence is as follows:

Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val

Pro Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Glu Glu Met

Gly Arg Gly Ser Met Ala Thr Pro Ser Met Met Pro Gln Trp Ala

Tyr Met His Ile Ala Gly Gln Asp Ala Ser Glu Tyr Leu Ser Pro

Gly Leu Val Gln Phe Ala Arg Ala Thr Asp Thr Tyr Phe Ser Met

Gly Asn Lys Phe Arg Asn Pro Thr Val Ala Pro Thr His Asp Val

Thr Thr Asp Arg Ser Gln Arg Leu Met Leu Arg Phe Val Pro Val

Asp Arg Glu Asp Asn Thr Tyr Ser Tyr Lys Val Arg Tyr Thr Leu

Ala Val Gly Asp Asn Arg Val Leu Asp Met Ala Ser Thr Phe Phe

Asp Ile Arg Gly Val Leu Asp Arg Gly Pro Ser Phe Lys Pro Tyr

Ser Gly Thr Ala Tyr Asn Ser Leu Ala Pro Lys Gly Ala Pro Asn

Thr Ser Gln Trp Gly Gly Ser Met Ala Thr Pro Ser Met Leu Pro

Gln Trp Ala Tyr Met His Ile Ala Gly Gln Asp Ala Ser Glu Tyr

Leu Ser Pro Gly Leu Val Gln Phe Ala Arg Ala Thr Asp Thr Tyr

Phe Asn Leu Gly Asn Lys Phe Arg Asn Pro Thr Val Ala Pro Thr

His Asp Val Thr Thr Asp Arg Ser Gln Arg Leu Met Leu Arg Phe

Val Pro Val Asp Arg Glu Asp Asn Thr Tyr Ser Tyr Lys Val Arg

Tyr Thr Leu Ala Val Gly Asp Asn Arg Val Leu Asp Met Ala Ser

Thr Phe Phe Asp Ile Arg Gly Val Leu Asp Arg Gly Pro Ser Phe

Lys Pro Tyr Ser Gly Thr Ala Tyr Asn Ser Leu Ala Pro Lys Gly

Ala Pro Asn Thr Ser Gln Trp Gly Gly Ser Met Ala Thr Pro Ser

Met Leu Pro Gln Trp Ala Tyr Met His Ile Ala Gly Gln Asp Ala

Ser Glu Tyr Leu Ser Pro Gly Leu Val Gln Phe Ala Arg Ala Thr

Asp Thr Tyr Phe Asn Leu Gly Asn Lys Phe Arg Asn Pro Thr Val

Ala Pro Thr His Asp Val Thr Thr Asp Arg Ser Gln Arg Leu Met

Leu Arg Phe Val Pro Val Asp Arg Glu Asp Asn Thr Tyr Ser Tyr

Lys Val Arg Tyr Thr Leu Ala Val Gly Asp Asn Arg Val Leu Asp

Met Ala Ser Thr Phe Phe Asp Ile Arg Gly Val Leu Asp Arg Gly

Pro Ser Phe Lys Pro Tyr Ser Gly Thr Ala Tyr Asn Ser Leu Ala

Pro Lys Gly Ala Pro Asn Thr Ser Gln Trp。

4. screening and identifying the engineering bacteria expressing the fusion protein:

the transformants were inoculated into a tube containing 3mL of LB medium (containing kanotoxin 60. mu.g/mL), cultured with shaking at 37 ℃ for 3 hours, IPTG was added to a final concentration of 0.5mmol/L, induction was continued for 4 hours, cells were collected by centrifugation and subjected to SDS-PAGE detection, and 6 transformants expressed adenovirus epitope fusion proteins having a relative molecular weight of 50kD (FIG. 4).

Purification of five serotype universal antigen epitope fusion proteins of human adenovirus

According to the amino acid sequence of five serotype universal antigen epitope fusion proteins of human adenovirus, the physicochemical properties of the five serotype universal antigen epitope fusion proteins are analyzed, and a proper purification method is determined. By computer analysis, the isoelectric point of the adenovirus epitope fusion protein was pH4.865, so we decided to purify it with a nickel ion affinity chromatography column in 1xPBS buffer at pH 7.4. The method comprises the following specific steps:

materials and methods

1. The main reagents are as follows:

Ni-NTA Resin is a product of Kinsley, and IPTG and DTT are products of TaTaRa. Other reagents are domestic or imported analytical pure reagents.

2. Induction expression and ultrasonic cracking of adenovirus epitope fusion protein engineering bacteria

A single colony is picked from an LB plate inoculated with the engineering bacterium No. 1 by using a toothpick, inoculated into a triangular flask containing 200mL of LB liquid culture medium, added with kanamycin to the final concentration of 50 mug/mL, and cultured in a shaking table at 37 ℃ overnight. The next day, the bacterial solution was inoculated into 4 Erlenmeyer flasks each containing 200mL of LB liquid medium, 50mL of bacterial solution was inoculated into each flask, the flasks were cultured in a shaker at 37 ℃ for 1 hour with shaking, and IPTG was added to a final concentration of 0.1mmol/L for induction expression for 4 hours.

1000mL of engineering bacteria for inducing expression of the fusion protein are centrifuged (8000 rpm, 30 min, 4 ℃) to collect the bacteria, the bacteria are suspended in 100mL of bacterial lysate (20 mmol/L PB pH7.4, 10 mmol/L EDTA, 1mmol/L DTT, 5% glycerol), the bacteria are broken by ultrasonic in an ice bath for 10min, and the precipitates are collected by centrifugation (8000 rpm, 30 min, 4 ℃).

3. Denaturation of adenovirus universal epitope fusion protein

Resuspending and washing the inclusion body by 30ml of inclusion body washing liquor, wherein the inclusion body washing liquor is 1xPBS pH7.4 and 1% Triton X-100, ultrasonically suspending the inclusion body under the ice bath condition until all precipitates are suspended, and centrifugally collecting the precipitated inclusion body. And (3) carrying out heavy suspension dissolution on the inclusion bodies by using 8 mol/L urea and 1xPBS (x-PbS) pH7.4 denaturing solution, and carrying out ultrasonic crushing under an ice bath condition until the inclusion bodies are completely dissolved. The supernatant was collected by centrifugation at 8000 rpm, 15min and 4 ℃.

4. Purifying by nickel column affinity chromatography

Washing Ni-NTA Resin with 1xPBS pH7.4, removing excessive 20% ethanol, filtering the supernatant collected by centrifugation with 0.22 μm filter membrane, removing impurities, mixing with Ni-NTA Resin to allow adenovirus universal epitope fusion protein to bind with Ni-NTA Resin, and stirring with glass rod for 1.5 h. Washing a chromatographic column with deionized water and a balance solution in sequence, loading the mixed solution on the column, carrying out balance cleaning with the balance solution with the volume being 7 times of the column volume, sequentially using elution proteins of 20, 40, 100, 200 and 500 mmol/L imidazole eluent containing 8 mol/L urea, collecting the elution peak proteins, detecting the peak proteins by 12% SDS-PAGE electrophoresis, and determining which elution peak contains the expressed adenovirus epitope fusion protein. The result shows that the peak of the imidazole elution protein of 100 mmol/L is the adenovirus epitope fusion protein.

5. Renaturation of adenovirus universal antigen epitope fusion protein

The denatured fusion protein obtained by purification is sequentially placed in PBS pH7.4 dialysate containing 6, 4, 3, 2, 1, 0 mol/L urea for sequential dialysis renaturation.

Results

SDS-PAGE analysis is carried out on proteins eluted from the nickel column by imidazole with different concentrations, and the electrophoresis result shows that the adenovirus universal epitope fusion protein exists in the peak of 100 mmol/L imidazole elution protein.

The peak of imidazole elution protein (100 mmol/L) was subjected to concentration by dialysis renaturation and SDS-PAGE to determine the concentration of the fusion protein, and it was found that the concentration of the fusion protein obtained by purification was 0.5mg/mL (FIG. 5).

Identification and application of five serotype universal antigen epitope fusion proteins of human adenovirus

The purified human adenovirus five serotype universal antigen epitope fusion protein is used as immunogen to immunize BALB/c mice to prepare antiserum. The experimental result shows that the fusion protein has good immunogenicity and antigenicity, and can be used for research of adenovirus vaccines or development of antibody detection reagents.

Materials and methods

1. Human adenovirus five serotype universal epitope fusion proteins: the invention is prepared.

2. Enzyme-linked reaction materials:

the enzyme linked plate is a 96-well plate produced by Shenzhen, and the goat anti-mouse IgG labeled by Horse Radish Peroxidase (HRP) is purchased from Sigma company. Other materials are conventional materials for enzyme-linked reactions.

2. Preparation of fusion protein antiserum:

the fusion protein is fully mixed and emulsified with Freund's adjuvant and aluminum adjuvant, and then injected into the abdominal cavity to immunize mice. Then, every two weeks, the fusion protein is mixed and emulsified with Freund's adjuvant and aluminum adjuvant, and then the booster immunization is carried out. A total of 4 immunizations were performed. Two weeks after the 4 th immunization, blood was collected from the orbit to prepare antiserum.

3. And (3) performing humoral immunity detection on the fusion protein:

and detecting the immunogenicity of the fusion protein by adopting an indirect ELISA method. The method comprises the following basic steps: diluting the fusion protein with CBS buffer solution (pH9.6) to a final concentration of 5 μ g/mL, coating 96-well enzyme label plate, incubating at 4 deg.C overnight at 100 μ L per well; then, the plate is fully washed by 1 per thousand PBST for 5 times, 10 percent of calf serum (FBS) is used as blocking liquid, 150 mu L of the blocking liquid is added into each hole, and the blocking liquid is blocked for 2 hours at 37 ℃; 1 per mill PBST is washed for 5 times, the fusion protein antiserum is diluted in a gradient manner (the dilution is 10 percent calf serum) according to the following proportion of 1:5000, 1:10000, 1:20000, 1:40000, 1:80000, 1:160000, 1:320000, 1:640000 and 100 mu L/hole, the incubation is carried out for 2 hours at 37 ℃, and the negative serum is diluted according to 1:10000 to be used as a control; fully washing the plate for 5 times by 1 per mill of PBST; adding 1: diluting goat anti-mouse IgG (diluted by 10% calf serum) at 5000 deg.C, 100 μ L/well, and incubating at 37 deg.C for 1 h; washing the plate for 5 times with 1 ‰ PBST, adding TMB color development solution, 50 μ L/hole, reacting at 37 deg.C in dark for 10 min; adding 1mol/L HCl, 50 muL/hole, and stopping the reaction; enzyme-linked immunosorbent assay (ELIASA) for determining each well A₄₅₀Value if P/N>2.1, the result can be judged to be positive.

4. Cell immunoassay of the fusion protein:

the secretion levels of four cytokines IL-2, IL-5, IL-10 and TNF-alpha in serum after mice are immunized are detected by adopting a double-antibody sandwich method. And sequentially adding diluted mouse serum (primary antibody) with a certain concentration into micropores coated with corresponding cytokine monoclonal antibodies of the mice, and combining with the HRP-labeled cytokine antibody to form an antibody-antigen-enzyme-labeled antibody compound. Enzyme-linked immunosorbent assay A₄₅₀The value is obtained.

5. Detection of antigenicity of the fusion protein:

the binding capacity of the outpatient serum and the adenovirus fusion protein is detected by an ELISA method. Coating the fusion protein on a 96-hole enzyme label plate by using CBS buffer solution, and respectively carrying out the following steps of 1:100 and 1: 1000. serum of a patient diluted by the proportion of 1:1000, 1:10000 and 1:1000000 is used as a primary antibody, goat anti-human IgM-HRP is used as a secondary antibody, and IgM in the serum of the patient infected by the five serotype adenoviruses is detected.

6. Development of the antibody detection test strip:

and coating the gold-labeled adenovirus fusion antigen and the gold-labeled rabbit IgG on a binding pad in a mixed manner, and coating the goat anti-human IgM and the goat anti-rabbit IgG on an NC membrane as a detection line (IgM) and a quality control line (C) respectively. Drying the reagent strip at room temperature, soaking in 5% fetal calf serum for sealing, sequentially adhering a glass cellulose membrane, an NC fiber membrane and a water absorbing material on a white plastic lining plate, assembling a colloidal gold test strip, detecting the serum of five serotype adenovirus infection patients and the serum of healthy human by using the assembled test strip, and simultaneously detecting 4 respiratory syncytial virus, mycoplasma pneumoniae, chlamydia pneumoniae, H3N2 influenza virus and other respiratory pathogen infection sera for comparison. Judging the specificity of the test strip according to the presence or absence of color development of the detection line

Results

1. Method for detecting humoral immunity effect of adenovirus universal antigen epitope fusion protein by indirect ELISA method

Diluting the purified adenovirus general antigen epitope fusion protein with CBS buffer solution to coat an enzyme label plate, and detecting antiserum obtained by immunizing a mouse with the adenovirus antigen epitope fusion protein. The result shows that the serum antibody titer of the universal epitope fusion protein of the human adenovirus is 1: 160000. the adenovirus epitope fusion protein can effectively stimulate an organism to generate an antibody after immunizing the organism, has a better humoral immunity effect, and lays a foundation for further developing adenovirus vaccines (figure 6).

2. Double-antibody sandwich method for detecting cell immune effect of adenovirus universal antigen epitope fusion protein

The cell immune effect of the fusion protein immunized mice is evaluated by measuring the cytokine level of the mice. And (3) taking the antiserum of the mice immunized for the fourth time, detecting the secretion condition of the cytokine by using a double-antibody sandwich method, and comparing and analyzing the secretion conditions of the cytokine in the sera of the mice of the aluminum adjuvant group and the Freund adjuvant group by using negative serum as a control. The aluminum adjuvant group can increase the secretion level of IL-5 and IL-10 compared with the negative serum (p<0.05). The Freund adjuvant group can improve IL-10 secretion level(p<0.05). The aluminum adjuvant group and the Freund adjuvant group have an effect of promoting the secretion of IL-10 (p<0.05), the promotion of IL-2 and TNF-alpha secretion level has no significant difference from negative serum ratio. The aluminum adjuvant group significantly promoted secretion of IL-5 than the Freund adjuvant group: (p<0.01). Compared with Freund's adjuvant, the aluminum adjuvant can stimulate the cellular immunity of mice better (FIG. 7).

3. Indirect ELISA method for detecting antigenicity of adenovirus general epitope fusion protein

The adenovirus fusion protein is used as a detection antigen, and the ELISA method is used for detecting the serum of five serotype adenovirus infected patients and the serum of healthy people. According to ELISA results, the adenovirus fusion protein can effectively detect IgM antibodies of adenovirus types 3, 7, 11, 14 and 55 in the serum of a patient. The fusion protein was found to have good antigenicity (FIG. 8).

4. Development of antibody detection test strip

The prepared fusion antigen is used as an antigen to develop a gold-labeled immunochromatography detection test strip, which is used for detecting the serum of five serotype adenovirus infected patients and the serum of healthy people, and simultaneously detecting the serum infected by four respiratory pathogens such as respiratory syncytial virus, mycoplasma pneumoniae, chlamydia pneumoniae, H3N2 influenza virus and the like for comparison. The results show that the developed test strip has reaction with the serum of five serotype adenovirus infected patients, and has no cross reaction with other four respiratory tract pathogen infected sera and normal human sera, which indicates that the developed IgM antibody detection test strip has better adenovirus antibody detection spectrum and better specificity (figure 9).

The sequence listing of five serotype universal antigen epitope fusion proteins of human adenovirus (HAdV) is shown in the attached document: a nucleotide or amino acid sequence table computer readable vector.

Sequence listing

<110> lie Wei

<120> human adenovirus five serotype universal antigen epitope chimeric protein and preparation and application thereof

<141> 2021-03-31

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 411

<212> PRT

<213> HAdv

<220>

<221> PEPTIDE

<222> (1)...(411)

<223> contains five serotypes of universal antigen epitope of human adenovirus.

<400> 1

Met Ala Thr Pro Ser Met Met Pro Gln Trp Ala Tyr Met His Ile Ala

1 5 10 15

Gly Gln Asp Ala Ser Glu Tyr Leu Ser Pro Gly Leu Val Gln Phe Ala

20 25 30

Arg Ala Thr Asp Thr Tyr Phe Ser Met Gly Asn Lys Phe Arg Asn Pro

35 40 45

Thr Val Ala Pro Thr His Asp Val Thr Thr Asp Arg Ser Gln Arg Leu

50 55 60

Met Leu Arg Phe Val Pro Val Asp Arg Glu Asp Asn Thr Tyr Ser Tyr

65 70 75 80

Lys Val Arg Tyr Thr Leu Ala Val Gly Asp Asn Arg Val Leu Asp Met

85 90 95

Ala Ser Thr Phe Phe Asp Ile Arg Gly Val Leu Asp Arg Gly Pro Ser

100 105 110

Phe Lys Pro Tyr Ser Gly Thr Ala Tyr Asn Ser Leu Ala Pro Lys Gly

115 120 125

Ala Pro Asn Thr Ser Gln Trp Gly Gly Ser Met Ala Thr Pro Ser Met

130 135 140

Leu Pro Gln Trp Ala Tyr Met His Ile Ala Gly Gln Asp Ala Ser Glu

145 150 155 160

Tyr Leu Ser Pro Gly Leu Val Gln Phe Ala Arg Ala Thr Asp Thr Tyr

165 170 175

Phe Asn Leu Gly Asn Lys Phe Arg Asn Pro Thr Val Ala Pro Thr His

180 185 190

Asp Val Thr Thr Asp Arg Ser Gln Arg Leu Met Leu Arg Phe Val Pro

195 200 205

Val Asp Arg Glu Asp Asn Thr Tyr Ser Tyr Lys Val Arg Tyr Thr Leu

210 215 220

Ala Val Gly Asp Asn Arg Val Leu Asp Met Ala Ser Thr Phe Phe Asp

225 230 235 240

Ile Arg Gly Val Leu Asp Arg Gly Pro Ser Phe Lys Pro Tyr Ser Gly

245 250 255

Thr Ala Tyr Asn Ser Leu Ala Pro Lys Gly Ala Pro Asn Thr Ser Gln

260 265 270

Trp Gly Gly Ser Met Ala Thr Pro Ser Met Leu Pro Gln Trp Ala Tyr

275 280 285

Met His Ile Ala Gly Gln Asp Ala Ser Glu Tyr Leu Ser Pro Gly Leu

290 295 300

Val Gln Phe Ala Arg Ala Thr Asp Thr Tyr Phe Asn Leu Gly Asn Lys

305 310 315 320

Phe Arg Asn Pro Thr Val Ala Pro Thr His Asp Val Thr Thr Asp Arg

325 330 335

Ser Gln Arg Leu Met Leu Arg Phe Val Pro Val Asp Arg Glu Asp Asn

340 345 350

Thr Tyr Ser Tyr Lys Val Arg Tyr Thr Leu Ala Val Gly Asp Asn Arg

355 360 365

Val Leu Asp Met Ala Ser Thr Phe Phe Asp Ile Arg Gly Val Leu Asp

370 375 380

Arg Gly Pro Ser Phe Lys Pro Tyr Ser Gly Thr Ala Tyr Asn Ser Leu

385 390 395 400

Ala Pro Lys Gly Ala Pro Asn Thr Ser Gln Trp

405 410

<210> 2

<211> 1242

<212> DNA

<213> HAdv

<220>

<221> CDS

<222> (1)...(1239)

<223> an artificially synthesized gene fragment encoding a chimeric protein containing epitopes common to five serotypes of human adenovirus.

<220>

<221> misc_feature

<222> (1240)...(1242)

<223> stop codon increased when synthesizing gene.

<400> 2

ggatccatgg ctacaccctc aatgatgcca caatgggcgt atatgcacat tgccggtcaa 60

gacgcgtccg aatacctctc tccgggtctg gttcaatttg ccagagccac cgacacctat 120

ttcagcatgg gtaataagtt ccgtaatccg acggttgcac cgacgcatga tgttaccaca 180

gatcgtagcc aacgtctgat gctgcgcttt gttccggtgg accgcgagga caacacctac 240

tcctacaagg tgcgttacac cctggcggtc ggtgacaacc gtgtcctgga tatggcgtct 300

acctttttcg atattcgtgg tgtacttgac cgcggaccga gcttcaaacc gtatagcggc 360

accgcataca acagcctggc gccgaaaggc gcaccaaata cgagccaatg gggtggcagc 420

atggctactc cgagcatgtt accgcagtgg gcgtatatgc acatcgcagg ccaggatgca 480

agcgaatatc tgtctccggg tctggtgcag ttcgcgcgtg ctaccgacac ctacttcaac 540

ctgggcaaca aattccgcaa cccgaccgtt gcgccgacgc acgacgtgac gaccgaccgt 600

tcccaacgtc tgatgctgcg tttcgtgccg gtcgatcgtg aggataacac gtactcgtac 660

aaagtacgtt ataccttggc tgtgggcgac aaccgtgttc tcgatatggc gagtaccttt 720

tttgatatcc gcggcgtatt ggaccgcggt ccgtctttca agccgtactc tggcactgcg 780

tataacagcc tggctccgaa gggtgcaccg aatacttcac agtggggcgg tagcatggcc 840

accccgagca tgctgcctca gtgggcatac atgcatatcg cgggtcagga tgcttcggag 900

tacttgagcc ctggtttggt tcagtttgcg cgggctaccg acacctattt taacctgggt 960

aataaatttc gtaatccgac cgttgcgccg acccatgatg tgaccaccga tcgatcccag 1020

cgtttgatgc tgcgtttcgt gccggttgac cgcgaagata acacttattc ctataaggtg 1080

cgctacaccc ttgcggttgg tgacaaccgc gtcctggaca tggcttccac ctttttcgac 1140

attcgtggcg tgttggacag aggcccgagt tttaagccgt atagcggtac ggcctacaat 1200

tccctggcgc caaaaggcgc cccaaatacg agccaatggt aa 1242

Claims (3)

1. The five serotype universal antigen epitope fusion protein of human adenovirus is formed by connecting adenovirus type 3, 7, 11, 14 and 55 antigen epitope protein fragments, wherein the protein fragments are connected by glycine and serine, the total length of the fusion protein is 411 amino acids, and the amino acid sequence is as follows:

Met Ala Thr Pro Ser Met Met Pro Gln Trp Ala Tyr Met His Ile Ala Gly Gln Asp Ala Ser Glu Tyr Leu Ser Pro Gly Leu Val Gln Phe Ala Arg Ala Thr Asp Thr Tyr Phe Ser Met Gly Asn Lys Phe Arg Asn Pro Thr Val Ala Pro Thr His Asp Val Thr Thr Asp Arg Ser Gln Arg Leu Met Leu Arg Phe Val Pro Val Asp Arg Glu Asp Asn Thr Tyr Ser Tyr Lys Val Arg Tyr Thr Leu Ala Val Gly Asp Asn Arg Val Leu Asp Met Ala Ser Thr Phe Phe Asp Ile Arg Gly Val Leu Asp Arg Gly Pro Ser Phe Lys Pro Tyr Ser Gly Thr Ala Tyr Asn Ser Leu Ala Pro Lys Gly Ala Pro Asn Thr Ser Gln Trp Gly Gly Ser Met Ala Thr Pro Ser Met Leu Pro Gln Trp Ala Tyr Met His Ile Ala Gly Gln Asp Ala Ser Glu Tyr Leu Ser Pro Gly Leu Val Gln Phe Ala Arg Ala Thr Asp Thr Tyr Phe Asn Leu Gly Asn Lys Phe Arg Asn Pro Thr Val Ala Pro Thr His Asp Val Thr Thr Asp Arg Ser Gln Arg Leu Met Leu Arg Phe Val Pro Val Asp Arg Glu Asp Asn Thr Tyr Ser Tyr Lys Val Arg Tyr Thr Leu Ala Val Gly Asp Asn Arg Val Leu Asp Met Ala Ser Thr Phe Phe Asp Ile Arg Gly Val Leu Asp Arg Gly Pro Ser Phe Lys Pro Tyr Ser Gly Thr Ala Tyr Asn Ser Leu Ala Pro Lys Gly Ala Pro Asn Thr Ser Gln Trp Gly Gly Ser Met Ala Thr Pro Ser Met Leu Pro Gln Trp Ala Tyr Met His Ile Ala Gly Gln Asp Ala Ser Glu Tyr Leu Ser Pro Gly Leu Val Gln Phe Ala Arg Ala Thr Asp Thr Tyr Phe Asn Leu Gly Asn Lys Phe Arg Asn Pro Thr Val Ala Pro Thr His Asp Val Thr Thr Asp Arg Ser Gln Arg Leu Met Leu Arg Phe Val Pro Val Asp Arg Glu Asp Asn Thr Tyr Ser Tyr Lys Val Arg Tyr Thr Leu Ala Val Gly Asp Asn Arg Val Leu Asp Met Ala Ser Thr Phe Phe Asp Ile Arg Gly Val Leu Asp Arg Gly Pro Ser Phe Lys Pro Tyr Ser Gly Thr Ala Tyr Asn Ser Leu Ala Pro Lys Gly Ala Pro Asn Thr Ser Gln Trp。

2. the five serotype general antigen epitope fusion protein of human adenovirus as claimed in claim 1, which is expressed and prepared by gene engineering technology, the specific method is as follows:

screening of adenovirus epitope protein fragments:

analyzing the amino acid sequences of human adenovirus type 3, 7, 11, 14 and 55 hexon proteins by using ANTHWIN and other software, screening out protein fragments with high homology in five serotype adenovirus hexon proteins, connecting each protein fragment by using two glycines and one serine, connecting in series to form a multi-epitope fusion protein, selecting codons preferred by prokaryotes, translating the fusion protein into corresponding nucleotide sequences, and adding a nucleotide sequence at the 5' end of the fusion protein gene fragmentBamH I cleavage site, 3' end added stop codon andXho i cleavage site, which makes it easy to clone the synthesized gene fragment into plasmid pET-28a +BamH I andXho i, chemically synthesizing the full-length gene sequence of the fusion protein in the enzyme cutting site;

human adenovirus type 3 epitope amino acid sequence:

Met Ala Thr Pro Ser Met Met Pro Gln Trp Ala Tyr Met His Ile Ala Gly Gln Asp Ala Ser Glu Tyr Leu Ser Pro Gly Leu Val Gln Phe Ala Arg Ala Thr Asp Thr Tyr Phe Ser Met Gly Asn Lys Phe Arg Asn Pro Thr Val Ala Pro Thr His Asp Val Thr Thr Asp Arg Ser Gln Arg Leu Met Leu Arg Phe Val Pro Val Asp Arg Glu Asp Asn Thr Tyr Ser Tyr Lys Val Arg Tyr Thr Leu Ala Val Gly Asp Asn Arg Val Leu Asp Met Ala Ser Thr Phe Phe Asp Ile Arg Gly Val Leu Asp Arg Gly Pro Ser Phe Lys Pro Tyr Ser Gly Thr Ala Tyr Asn Ser Leu Ala Pro Lys Gly Ala Pro Asn Thr Ser Gln Trp；

human adenovirus type 7 epitope amino acid sequence:

Met Ala Thr Pro Ser Met Met Pro Gln Trp Ala Tyr Met His Ile Ala Gly Gln Asp Ala Ser Glu Tyr Leu Ser Pro Gly Leu Val Gln Phe Ala Arg Ala Thr Asp Thr Tyr Phe Ser Met Gly Asn Lys Phe Arg Asn Pro Thr Val Ala Pro Thr His Asp Val Thr Thr Asp Arg Ser Gln Arg Leu Met Leu Arg Phe Val Pro Val Asp Arg Glu Asp Asn Thr Tyr Ser Tyr Lys Val Arg Tyr Thr Leu Ala Val Gly Asp Asn Arg Val Leu Asp Met Ala Ser Thr Phe Phe Asp Ile Arg Gly Val Leu Asp Arg Gly Pro Ser Phe Lys Pro Tyr Ser Gly Thr Ala Tyr Asn Ser Leu Ala Pro Lys Gly Ala Pro Asn Thr Ser Gln Trp；

human adenovirus type 11 epitope amino acid sequence:

Met Ala Thr Pro Ser Met Leu Pro Gln Trp Ala Tyr Met His Ile Ala Gly Gln Asp Ala Ser Glu Tyr Leu Ser Pro Gly Leu Val Gln Phe Ala Arg Ala Thr Asp Thr Tyr Phe Asn Leu Gly Asn Lys Phe Arg Asn Pro Thr Val Ala Pro Thr His Asp Val Thr Thr Asp Arg Ser Gln Arg Leu Met Leu Arg Phe Val Pro Val Asp Arg Glu Asp Asn Thr Tyr Ser Tyr Lys Val Arg Tyr Thr Leu Ala Val Gly Asp Asn Arg Val Leu Asp Met Ala Ser Thr Phe Phe Asp Ile Arg Gly Val Leu Asp Arg Gly Pro Ser Phe Lys Pro Tyr Ser Gly Thr Ala Tyr Asn Ser Leu Ala Pro Lys Gly Ala Pro Asn Thr Ser Gln Trp；

human adenovirus type 14 epitope amino acid sequence:

Met Ala Thr Pro Ser Met Leu Pro Gln Trp Ala Tyr Met His Ile Ala Gly Gln Asp Ala Ser Glu Tyr Leu Ser Pro Gly Leu Val Gln Phe Ala Arg Ala Thr Asp Thr Tyr Phe Asn Leu Gly Asn Lys Phe Arg Asn Pro Thr Val Ala Pro Thr His Asp Val Thr Thr Asp Arg Ser Gln Arg Leu Met Leu Arg Phe Val Pro Val Asp Arg Glu Asp Asn Thr Tyr Ser Tyr Lys Val Arg Tyr Thr Leu Ala Val Gly Asp Asn Arg Val Leu Asp Met Ala Ser Thr Phe Phe Asp Ile Arg Gly Val Leu Asp Arg Gly Pro Ser Phe Lys Pro Tyr Ser Gly Thr Ala Tyr Asn Ser Leu Ala Pro Lys Gly Ala Pro Asn Ala Ser Gln Trp；

human adenovirus type 55 epitope amino acid sequence:

Met Ala Thr Pro Ser Met Leu Pro Gln Trp Ala Tyr Met His Ile Ala Gly Gln Asp Ala Ser Glu Tyr Leu Ser Pro Gly Leu Val Gln Phe Ala Arg Ala Thr Asp Thr Tyr Phe Asn Leu Gly Asn Lys Phe Arg Asn Pro Thr Val Ala Pro Thr His Asp Val Thr Thr Asp Arg Ser Gln Arg Leu Met Leu Arg Phe Val Pro Val Asp Arg Glu Asp Asn Thr Tyr Ser Tyr Lys Val Arg Tyr Thr Leu Ala Val Gly Asp Asn Arg Val Leu Asp Met Ala Ser Thr Phe Phe Asp Ile Arg Gly Val Leu Asp Arg Gly Pro Ser Phe Lys Pro Tyr Ser Gly Thr Ala Tyr Asn Ser Leu Ala Pro Lys Gly Ala Pro Asn Thr Ser Gln Trp；

fusion protein amino acid sequence:

Met Ala Thr Pro Ser Met Met Pro Gln Trp Ala Tyr Met His Ile Ala Gly Gln Asp Ala Ser Glu Tyr Leu Ser Pro Gly Leu Val Gln Phe Ala Arg Ala Thr Asp Thr Tyr Phe Ser Met Gly Asn Lys Phe Arg Asn Pro Thr Val Ala Pro Thr His Asp Val Thr Thr Asp Arg Ser Gln Arg Leu Met Leu Arg Phe Val Pro Val Asp Arg Glu Asp Asn Thr Tyr Ser Tyr Lys Val Arg Tyr Thr Leu Ala Val Gly Asp Asn Arg Val Leu Asp Met Ala Ser Thr Phe Phe Asp Ile Arg Gly Val Leu Asp Arg Gly Pro Ser Phe Lys Pro Tyr Ser Gly Thr Ala Tyr Asn Ser Leu Ala Pro Lys Gly Ala Pro Asn Thr Ser Gln Trp Gly Gly Ser Met Ala Thr Pro Ser Met Leu Pro Gln Trp Ala Tyr Met His Ile Ala Gly Gln Asp Ala Ser Glu Tyr Leu Ser Pro Gly Leu Val Gln Phe Ala Arg Ala Thr Asp Thr Tyr Phe Asn Leu Gly Asn Lys Phe Arg Asn Pro Thr Val Ala Pro Thr His Asp Val Thr Thr Asp Arg Ser Gln Arg Leu Met Leu Arg Phe Val Pro Val Asp Arg Glu Asp Asn Thr Tyr Ser Tyr Lys Val Arg Tyr Thr Leu Ala Val Gly Asp Asn Arg Val Leu Asp Met Ala Ser Thr Phe Phe Asp Ile Arg Gly Val Leu Asp Arg Gly Pro Ser Phe Lys Pro Tyr Ser Gly Thr Ala Tyr Asn Ser Leu Ala Pro Lys Gly Ala Pro Asn Ala Ser Gln Trp Gly Gly Ser Met Ala Thr Pro Ser Met Leu Pro Gln Trp Ala Tyr Met His Ile Ala Gly Gln Asp Ala Ser Glu Tyr Leu Ser Pro Gly Leu Val Gln Phe Ala Arg Ala Thr Asp Thr Tyr Phe Asn Leu Gly Asn Lys Phe Arg Asn Pro Thr Val Ala Pro Thr His Asp Val Thr Thr Asp Arg Ser Gln Arg Leu Met Leu Arg Phe Val Pro Val Asp Arg Glu Asp Asn Thr Tyr Ser Tyr Lys Val Arg Tyr Thr Leu Ala Val Gly Asp Asn Arg Val Leu Asp Met Ala Ser Thr Phe Phe Asp Ile Arg Gly Val Leu Asp Arg Gly Pro Ser Phe Lys Pro Tyr Ser Gly Thr Ala Tyr Asn Ser Leu Ala Pro Lys Gly Ala Pro Asn Thr Ser Gln Trp；

DNA sequence of chemically synthesized fusion protein

GGA TCC ATG GCT ACA CCC TCA ATG ATG CCA CAA TGG GCG TAT ATG CAC ATT GCC GGT CAA GAC GCG TCC GAA TAC CTC TCT CCG GGT CTG GTT CAA TTT GCC AGA GCC ACC GAC ACC TAT TTC AGC ATG GGT AAT AAG TTC CGT AAT CCG ACG GTT GCA CCG ACG CAT GAT GTT ACC ACA GAT CGT AGC CAA CGT CTG ATG CTG CGC TTT GTT CCG GTG GAC CGC GAG GAC AAC ACC TAC TCC TAC AAG GTG CGT TAC ACC CTG GCG GTC GGT GAC AAC CGT GTC CTG GAT ATG GCG TCT ACC TTT TTC GAT ATT CGT GGT GTA CTT GAC CGC GGA CCG AGC TTC AAA CCG TAT AGC GGC ACC GCA TAC AAC AGC CTG GCG CCG AAA GGC GCA CCA AAT ACG AGC CAA TGG GGT GGC AGC ATG GCT ACT CCG AGC ATG TTA CCG CAG TGG GCG TAT ATG CAC ATC GCA GGC CAG GAT GCA AGC GAA TAT CTG TCT CCG GGT CTG GTG CAG TTC GCG CGT GCT ACC GAC ACC TAC TTC AAC CTG GGC AAC AAA TTC CGC AAC CCG ACC GTT GCG CCG ACG CAC GAC GTG ACG ACC GAC CGT TCC CAA CGT CTG ATG CTG CGT TTC GTG CCG GTC GAT CGT GAG GAT AAC ACG TAC TCG TAC AAA GTA CGT TAT ACC TTG GCT GTG GGC GAC AAC CGT GTT CTC GAT ATG GCG AGT ACC TTT TTT GAT ATC CGC GGC GTA TTG GAC CGC GGT CCG TCT TTC AAG CCG TAC TCT GGC ACT GCG TAT AAC AGC CTG GCT CCG AAG GGT GCA CCG AAT ACT TCA CAG TGG GGC GGT AGC ATG GCC ACC CCG AGC ATG CTG CCT CAG TGG GCA TAC ATG CAT ATC GCG GGT CAG GAT GCT TCG GAG TAC TTG AGC CCT GGT TTG GTT CAG TTT GCG CGG GCT ACC GAC ACC TAT TTT AAC CTG GGT AAT AAA TTT CGT AAT CCG ACC GTT GCG CCG ACC CAT GAT GTG ACC ACC GAT CGA TCC CAG CGT TTG ATG CTG CGT TTC GTG CCG GTT GAC CGC GAA GAT AAC ACT TAT TCC TAT AAG GTG CGC TAC ACC CTT GCG GTT GGT GAC AAC CGC GTC CTG GAC ATG GCT TCC ACC TTT TTC GAC ATT CGT GGC GTG TTG GAC AGA GGC CCG AGT TTT AAG CCG TAT AGC GGT ACG GCC TAC AAT TCC CTG GCG CCA AAA GGC GCC CCA AAT GCA AGC CAA TGG TAA CTC GAG；

Construction of five serotype universal antigen epitope fusion protein recombinant plasmids of human adenovirus:

extracting plasmid pET-28a (+) byBamH I and Xho IDouble digestion, recovery of digested plasmid large fragment after electrophoresis, dissolving in deionized water, and useBamH I and Xho IHuman adenovirus five serotype general antigen epitope fusion protein gene fragment synthesized by double enzyme digestion chemistry, after electrophoretic recovery, dissolved in deionized water, and the DNA fragment after enzyme digestion is taken and inserted into a carrier pET28a (+)BamH I and Xho IAmong the sites, the fusion protein containing five serotype adenovirus antigen epitopes is expressed;

screening and identifying recombinant plasmids:

the recombinant plasmid was transformed into E.coli BL21(DE3), coated with a kanamycin-containing LB plate, and left overnight at 37 ℃; randomly selecting transformed colony and control bacteria, plasmid pET-28a transformed bacteria, extracting plasmid, and usingBamH I and Xho IDouble enzyme digestion verification, wherein a 1.0% agarose gel electrophoresis result shows that a 1248bp target gene fragment is cut; at the same time, DNA sequence analysis is carried out on the plasmid containing the gene fragmentSequence analysis proves that the recombinant plasmid contains five serotype adenovirus epitope gene fragments which are connected together, and the sequence is completely correct;

the constructed recombinant plasmid expresses fusion protein containing five serotype adenovirus antigen epitopes, the total length is 445 amino acids, the first 135 amino acid fragments of five serotype adenovirus hexon proteins are connected by two glycines and one serine, and the amino acid sequences are as follows:

Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val

Pro Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Glu Glu Met

Gly Arg Gly Ser Met Ala Thr Pro Ser Met Met Pro Gln Trp Ala

Tyr Met His Ile Ala Gly Gln Asp Ala Ser Glu Tyr Leu Ser Pro

Gly Leu Val Gln Phe Ala Arg Ala Thr Asp Thr Tyr Phe Ser Met

Gly Asn Lys Phe Arg Asn Pro Thr Val Ala Pro Thr His Asp Val

Thr Thr Asp Arg Ser Gln Arg Leu Met Leu Arg Phe Val Pro Val

Asp Arg Glu Asp Asn Thr Tyr Ser Tyr Lys Val Arg Tyr Thr Leu

Ala Val Gly Asp Asn Arg Val Leu Asp Met Ala Ser Thr Phe Phe

Asp Ile Arg Gly Val Leu Asp Arg Gly Pro Ser Phe Lys Pro Tyr

Ser Gly Thr Ala Tyr Asn Ser Leu Ala Pro Lys Gly Ala Pro Asn

Thr Ser Gln Trp Gly Gly Ser Met Ala Thr Pro Ser Met Leu Pro

Gln Trp Ala Tyr Met His Ile Ala Gly Gln Asp Ala Ser Glu Tyr

Leu Ser Pro Gly Leu Val Gln Phe Ala Arg Ala Thr Asp Thr Tyr

Phe Asn Leu Gly Asn Lys Phe Arg Asn Pro Thr Val Ala Pro Thr

His Asp Val Thr Thr Asp Arg Ser Gln Arg Leu Met Leu Arg Phe

Val Pro Val Asp Arg Glu Asp Asn Thr Tyr Ser Tyr Lys Val Arg

Tyr Thr Leu Ala Val Gly Asp Asn Arg Val Leu Asp Met Ala Ser

Thr Phe Phe Asp Ile Arg Gly Val Leu Asp Arg Gly Pro Ser Phe

Lys Pro Tyr Ser Gly Thr Ala Tyr Asn Ser Leu Ala Pro Lys Gly

Ala Pro Asn Thr Ser Gln Trp Gly Gly Ser Met Ala Thr Pro Ser

Met Leu Pro Gln Trp Ala Tyr Met His Ile Ala Gly Gln Asp Ala

Ser Glu Tyr Leu Ser Pro Gly Leu Val Gln Phe Ala Arg Ala Thr

Asp Thr Tyr Phe Asn Leu Gly Asn Lys Phe Arg Asn Pro Thr Val

Ala Pro Thr His Asp Val Thr Thr Asp Arg Ser Gln Arg Leu Met

Leu Arg Phe Val Pro Val Asp Arg Glu Asp Asn Thr Tyr Ser Tyr

Lys Val Arg Tyr Thr Leu Ala Val Gly Asp Asn Arg Val Leu Asp

Met Ala Ser Thr Phe Phe Asp Ile Arg Gly Val Leu Asp Arg Gly

Pro Ser Phe Lys Pro Tyr Ser Gly Thr Ala Tyr Asn Ser Leu Ala

Pro Lys Gly Ala Pro Asn Thr Ser Gln Trp；

screening and identifying the engineering bacteria expressing the fusion protein:

inoculating the positive transformant containing the recombinant plasmid into a test tube containing 3mL of LB culture medium containing 50 mu g/mL of kanotoxin, carrying out shake culture at 37 ℃ for 2h, adding IPTG (isopropyl-beta-thiogalactoside) to a final concentration of 0.5mmol/L, continuing to carry out shake culture induction for 4h, centrifugally collecting thalli, carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) detection, and expressing the recombinant plasmid with a relative molecular weight of 50kD

Five serotypes of human adenovirus universal antigen epitope fusion protein, while the control bacterium BL21(DE3) has no protein band;

purification of five serotype universal antigen epitope fusion proteins of human adenovirus:

1) ultrasonic lysis of five serotype universal antigen epitope fusion protein engineering bacteria of human adenovirus

Centrifuging engineering bacteria for inducing expression of the fusion protein (8000 rpm, 15min and 4 ℃) to collect bacteria, suspending the bacteria in bacterial lysate (20 mmol/L PB pH7.4 and 5% glycerol) with the volume of 1/10 of original culture solution, ultrasonically breaking the bacteria in ice bath, centrifuging and collecting precipitates, wherein the precipitates are expressed adenovirus epitope fusion protein inclusion bodies;

2) denaturation of five serotype universal epitope fusion proteins of human adenovirus

Resuspending and washing the inclusion body by 30ml of inclusion body washing liquor, wherein the inclusion body washing liquor is 1xPBS pH7.4 and 1% Triton X-100, ultrasonically suspending the inclusion body under an ice bath condition until all precipitates are suspended, centrifugally collecting the precipitated inclusion body, resuspending and dissolving the inclusion body by using 8 mol/L urea and 1xPBS pH7.4 denaturing solution, ultrasonically crushing the inclusion body under the ice bath condition until the inclusion body is completely dissolved, and centrifugally collecting supernatant at 8000 rpm, 15min and 4 ℃;

3) purifying by nickel column affinity chromatography

Washing Ni-NTA Resin with 1xPBS pH7.4, removing excessive 20% ethanol, filtering the supernatant collected by centrifugation with 0.22 μm filter membrane, removing impurities, mixing with Ni-NTA Resin, allowing human adenovirus five serotype universal epitope fusion protein to bind with Ni-NTA Resin, stirring with glass rod for 1.5h, washing the chromatographic column with deionized water and balance solution in sequence, then loading the mixed solution into a column, carrying out balanced cleaning by using a balanced solution with 7 times of column volume, sequentially using elution proteins of 20, 40, 100, 200 and 500 mmol/L imidazole eluent containing 8 mol/L urea, collecting each elution peak protein, detecting each peak protein by using 12% SDS-PAGE electrophoresis, determining which elution peak contains the expressed adenovirus epitope fusion protein, and displaying the result that the 100 mmol/L imidazole elution protein peak is the adenovirus epitope fusion protein;

renaturation of five serotype universal antigen epitope fusion protein of human adenovirus

The denatured fusion protein obtained by purification is sequentially placed in PBS pH7.4 dialysate containing 6, 4, 3, 2, 1, 0 mol/L urea for sequential dialysis renaturation.

3. The use of the five serotype universal epitope fusion proteins of human adenovirus according to claim 1 for the preparation of adenovirus vaccines, antibodies or antigen detection reagents.

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